A TECHNICAL REPORT ON

STUDENT INDUSTRIAL WORK EXPERIENCE SCHEME (SIWES)

AT:

CENTRE FOR DRYLAND AGRICULTURE

BAYERO UNIVERSITY, KANO STATE.

BY

YASIN TIJJANI ABDURRAHMAN

(FSC/BOT/15/0003)

SUBMITTED TO:

THE DEPARTMENT OF BIOLOGICAL SCIENCES FEDERAL UNIVERSITY

DUTSE, JIGAWA STATE.

IN PARTIAL FULFILLMENT FOR THE AWARD OF BACHELOR OF SCIENCE

(B.Sc) DEGREE IN BOTANY.

AUGUST, 2018.

i
 A Technical Report on

Student Industrial Work Experience Scheme (SIWES)

ii
 DEDICATION

I dedicated this study and write up to my lovely parents; Hajiya Khadijat & Alhaji Tijjani

Abdurrahman for their love and their unforgetful support, may Allah reward them with

Jannatul Firdaus, Ameen.

iii
 ACKNOWLEDGEMENT

All thanks and praises be to almighty Allah (S.W.T) whose infinite mercy brought me to the

completion of this programme, May the beneficent and salutation of Allah be upon his

Prophet Muhammad (PBUH).

I would like to acknowledge the complementary effort of my parents and my siblings for

their support morally and financially towards the completion of this work and also my entire

friends and family for their incessant support.

I wish to convey my sincere gratitude to my industrial based coordinator Dr. Kabir

Mustapha Umar. And also my industrial based supervisors Miss Mary Olukoredo and Malam

Adam M. Adam for taking their time to assist and guide me in carrying out most of the

training. Also to my institution based SIWES coordinator Malam D. A. Sufi and my

supervisor Dr. Maryam Muhammad, who in her busy schedule still finds the time to come for

my supervision.

iv
 CERTIFICATION

This is to certify that Yasin Tijjani Abdurrahman has done his student industrial work

experience (SIWES) at the Centre for Dryland Agriculture, Bayero University, Kano.

_________________ Date: ____________

Yasin Tijjani Abdurrahman

__________________ Date: ____________

Supervisor;

Dr. Maryam Muhammad

____________________ Date: _____________

Siwes Coordinator

Malam D. A. Sufi

v
 ABSTRACT

The Centre for Dryland Agriculture (CDA) is a regional centre of excellence established to

produce highly skilled manpower and research products that will address the challenges of

agricultural development within the dryland areas of West and Central Africa (WCA) and

expand the capacity of the region to deal with issues of food security, natural resources

management, climate change, livelihoods and broader development challenges.

Various activities have been carried out during the course of the programm. Tissue Culture,

Wet Chemistry, and Central Laboratory were the three laboratories where the training was

conducted. In tissue culture laboratory, general introduction to Tissue Culture, preparation for

seed and organ culture were carried out. In the wet chemistry laboratory, soil physical and

chemical properties were analyzed using standard analytical procedures. Lastly, in the central

laboratory preparation of standards, centrifugation and elemental analysis were carried out.

vi
 TABLE OF CONTENTS

COVER PAGE ……………………………………………………………………………....i

TITLE PAGE………………………………………………………………………………… ii

DEDICATION……………………………………………………………………………..... iii

ACKNOWLEDGEMENT…………………………………………………………………... iv

CERTIFICATION……………………………………………………………………….…… v

ABSTRACT…………………………………………………………………………………. vi

TABLE OF CONTENTS ……………………………………………………………………vii

CHAPTER ONE ……………………………………………………………………………...1

INTRODUCTION……………………………………………………………………………..1

1.1 INTRODUCTION…………………………………………………………………………1

1.2 STUDENT INDUSTRIAL WORK EXPERIENCE SCHEME (SIWES)

BACKGROUND………………………………………………………………………………2

1.2.1 GROWTH OF THE SCHEME ……………….………………………………………...4

1.3 OBJECTIVES of SIWES …………………………………………………………………4

1.3.1 THE ROLE OF STUDENTS AND INSTITUTION IN SIWES ……………………….5

1.4 BODIES INVOLVED IN THE MANAGEMENT OF SIWES ……………….………….5

1.4.1 ROLES PLAYED BY THE BODIES ………………………………...………………...5

vii
CHAPTER TWO ………………………………………………………………...……………7

BACKGROUND OF COMPANY/ORGANIZATION ………………………………….…...7

2.1 INTRODUCTION ……………………………………………………...…………………7

2.2 HISTORY ……………………………………………………………...………………….8

2.3 ORGANOGRAM …………………………………………………..……………………10

2.4 VISION AND MISSION …………………………………………..……………………11

CHAPTER THREE …………………………………………………….……………………12

THE PROCESS, COMPONENTS & DESCRIPTION …………….……………………..…12

3.1 INTRODUCTION ……………………………………………….………………………12

3.2 TISSUE CULTURE LABORATORY …………………………..………………………12

3.3 WET CHEMISTRY LABORATORY ………………………….……………………….13

3.4 CENTRAL LABORATORY (INSTRUMENTATION) ………..………………………14

CHAPTER FOUR …………………………………………………..……………………….15

WORKING EXPERIENCE …………………………………..……………………………..15

4.1 INRODUCTION …………………………………………..……………………………15

4.2 TISSUE CULTURE ……………………………………………………………………..15

4.2.1 TYPES OF PLANT TISSUE CULTURE ………………………………………..........15

4.2.2 PHASESOF PLANT TISSUE CULTURE ……………………………………………18

4.2.3 BASIC REQUIREMENTS OF PLANT TISSUE CULTURE …………………..........19

viii
4.2.4 PREPARATION AND STERILIZATION OF CULTURE MEDIA …………………21

4.2.5 GENARAL TECHNIQUES OF PLANT TISSUE CULTURE…………………..........24

4.3 WET CHEMISTRY ……………………………………………………………………..30

4.3.1 SOIL pH AND ELECTROCONDUCTIVITY …………………………………..........31

4.3.2 SOIL PARTICLE SIZE ANALYSIS ………………………………………………….32

4.3.3 ORGANIC CARBON CONTENT OF SOILS …………………………………..........34

4.3.4 MEHLICH-3 EXTRACTION AND ANALYSIS ……………………………………35

4.4 CENTRAL LABORATORY (INSTRUMENTATION) ……………………...……...…36

4.4.1 DETERMINATION OF MACRO AND MICRONUTRIENTS IN PLANTS …..........36

4.5 PROBLEMS ENCOUNTERED ………………………………………………………...37

4.6 PROBLEMS SOLVED ………………………………………………………………….38

CHAPTER FIVE …………………………………………………………………………….39

SUMMARY, RECOMMENDATION AND CONCLUSION ……………………………...39

5.1 INTRODUCTION ……………………………………………………………………….39

5.2 SUMMARY ……………………………………………………………………………..39

5.3 RECOMMENDATION …………………………………………………………………40

5.4 CONCLUSION ………………………………………………………………………….40

REFERENCES ………………………………………………………………………………42

ix
 CHAPTER ONE

INTRODUCTION

1.1 INTRODUCTION

The Student Industrial Work Experience Scheme (SIWES) was initiated in the year 1973 by

the Industrial Training Fund and set up via the decree No. 47 of 1971 by the Federal

Government to fill the gap between theories thought in tertiary institutions and practical

aspect being acquired in the industries.

Training is a key factor in enhancing the efficiency and experience of the Work force.

The Student Industrial Work experience scheme (SIWES) program prepare student for labour

markets, it has become an innovative phenomenon in human resources development and

training in Nigeria.

Practical knowledge recites to doing according to Chinghai (1995): practical knowledge is

learning without which mastery of an area of knowledge may be too difficult to achieve,

practical knowledge involves developing skills through the use of tools or equipment to

perform task that are related to a field of study.

No society can achieve meaningful progress without encouraging its youth to acquire

compulsory practical skills. Such skills help them to harness available resources to meet the

need of society.

It was against this background that SIWES, otherwise referred to as Industrial Training (IT),

was introduced in Nigerian tertiary institution.

Technical education exist to serve industries, It is therefore necessary that a high degree of

cooperation to be maintained between Technical and industries.

1
The forth common wealth education recommended that industries should be closely

associated with the technological education in curriculum development Provision of

opportunities for Industrial experience accreditation, consultancy services, part time course

and vocational guidance cooperation between in situation.

The Industrial attachment program fulfils part of the requirement in pursuing the degree of

Bachelor of Science in Botany in Federal University Dutse. This report services to summarize

the activities and experiences I acquire at CENTRE FOR DRYLAND AGRICULTURE,

Bayero University (BUK) kano.

1.2 STUDENT INDUSTRIAL WORK EXPERIENCE SCHEME (SIWES)

BACKGROUND

The Student Industrial Work Experience Scheme (SIWES) is an accepted skills programme

which forms part of the approved academic standards in the degree programme for Nigerian

Universities. In 1974, the Federal Government of Nigeria introduced the national policy on

Industrial training, called the Students, Industrial Work Experience Scheme (SIWES). This

programme is under the umbrella of the Ministry of Education through the Industrial Training

Fund (ITF), designed to help students acquire the necessary practical education/experience in

their fields of study and other related professions.

The government decree No. 47 of 8th October, 1971 as amended in 1990 Highlights to

capacity building of human resources in industry commerce and government through training

and retraining of workers in order to effectively provide the much needed high quality goods

and services in a dynamic economy as ours. This decree led to the establishment of industrial

training fund. The growing concern among our industrialist that graduates of our institutions

of higher learning lack adequate practical background studies preparatory for employment in

industries Led to the formation of student industrial work experience scheme (SIWES) by

2
ITF in 1993/1994. ITF has as one cooperative entity with industry and commerce where

students in institutions of higher learning can undertake mid carrier work experience

attachments in industries which are capable with student's area of study.

This is an effort which was created in order to bridge the existing gap between the theory

taught in the classroom and practice of science, agriculture, medicine, engineering,

technology and other professional programmes in the Nigerian tertiary institutions. This

programme is aimed at exposing the students to the use of various machines and equipments,

professional work methods and ways of safeguarding the work areas in industries as well as

other organizations and parastatal. The programme was established basically to impact

elaborate practical understanding to students with respect to their various disciplines. It is

also intended that the student through a process of relation to academic knowledge and

practical Industrial application would understand the underlying principles and become better

focused and acquire the practical applications towards excellence in his or her discipline.

The Students Industrial Work Experience Scheme (SIWES) programme involves the student,

the Universities and the industries. This training is funded by the Federal Government of

Nigeria and jointly coordinated by the Industrial Training Fund (ITF) and the National

Universities Commission (NUC).This framing program is undertaken in the third year of a

four year degree program, the minimum duration of the program is normally 24 weeks except

for engineering and technological programs where the minimum duration is 40 weeks.

Industrial Training Fund (ITF) was established in 1971, It has operated consistently and

painstakingly within the context of its enabling laws Decree 47 of 1971 as amended in the

2011 ITF ACT. Following established of ITF, SIWES commanded in 1974 with the

Following aims and objectives.

3
1.2.1 GROWTH OF THE SCHEME

Since the inception of SIWES, the scope of the scheme has widened considerably. The

number of institution grew up with its corresponding increase in the number of student's

population. At inception, only 11 programs were part of the scheme with 784 students

enrolled. At present there are over 100 programs in the scheme with over 200,000 thousand

student enrolled.

1.3 OBJECTIVES OF SIWES

SIWES provides avenue for student to acquire industrial skills and experience in their

approved course of study. It also prepare student for their industrial work situation after

graduation.

The objectives of the student’s industrial training work experience scheme are:

1. Provision of avenue for students in the Nigerian universities to gain Industrial skills

and experience in their course of study.

2. To prepare students for the work situation they are likely to meet after graduation.

3. To expose students to work methods and techniques in handling equipment and

machinery that may not be available in the universities.

4. To make the transition from the University to the world of world of work easier, and

thus enhance students contacts for later job placement.

5. To provide students with an opportunity to apply their theoretical knowledge in real

work situation, thereby bridging the gap between university work and actual practice.

6. To enlist and strengthen employers involvement in entire educational process of

preparing university graduates for employment.

4
1.3.1 THE ROLE OF STUDENTS AND INSTITUTION IN SIWES

The role of students is to partake in the program in such a way that they will achieve

maximum benefit from the program. Also give student a great chance to ask questions, be

submissive and adhere to all rules and regulations of the organization where they are

attached. While identification of placement opportunities, funding of SIWES supervision and

assessment of the student are some of the roles played by the institution to ensure smooth

running of the program.

1.4 BODIES INVOLVED IN THE MANAGEMENT OF SIWES

The bodies involved in the management of SIWES:

 The Federal Government

 The Industrial Training Fund (ITF)

 The Supervising Agencies

 National Universities Commission (NUC)

 National Board for Technical Education (NBTE)

 National Commission for Colleges of Education (NCCE)

1.4.1 ROLES PLAYED BY THE BODIES

1. Federal Government:

 To make adequate funds available to the Federal Ministry of Industry to fund the

scheme.

 Make it mandatory for all ministries, companies and government parastatals, to offer

an attachment places to students.

5
  Make it a policy include a clause in every major contact lasting over six to nine

months begin awards for contractors to take in students in attachment.

2. The Industrial Training Fund (ITF):

 To provide logistic material needed to administer the scheme.

 Compile a list of employers and available training places for industrial attachment and

forward such list to the coordinating agencies (i.e. NUC, NBTE, NCCE).

 Organize conferences and famine on SIWES.

3. The supervising agencies (NUC, NBTE, NCCE)

4. These agencies are to:

I. Ensure the establishment and accreditation of SIWES Units in institutions under

their jurisdiction.

II. Ensure adequate Funding of the SIWES in all the institution.

III. Continuously monitor and review the Job specification of all the coyness

6
 CHAPTER TWO

BACKGROUND OF COMPANY/ORGANIZATION

2.1 INTRODUCTION

The Centre for Dryland Agriculture (CDA) is a regional centre of excellence established to

produce highly skilled manpower and research products that will address the challenges of

agricultural development within the dryland areas of West and Central Africa (WCA) and

expand the capacity of the region to deal with issues of food security, natural resources

management, climate change, livelihoods and broader development challenges. Situated in

Bayero University, Kano (BUK), the centre was formally established in 2012 through a

competitively won take-off grant from MacAthur foundation. The CDA is one of Africa

Centres of Excellence (ACE) supported by the World Bank, the association of African

Universities (AAU) and Nigeria’s National Universities Commission (NUC). This support

has enabled the centre to build and install excellent teaching, learning and research facilities

geared towards building capacities for a wide range of stakeholders, expanding national and

regional outreach, supporting innovative research and upgrading the teaching and research

capacities within the WCA sub-region. Founded on the tripod of teaching, research and

outreach, a cardinal objective of the CDA is the transformation of teaching methodologies,

redefining the postgraduate teaching curriculum, engaging broad level stakeholders and

building capacities that will lead to the achievement of improved agricultural production and

livelihoods and the sustainable management of natural resources. The Centre has gradually

become a household name among professionals and practitioners not only in Nigeria, but the

entire West Africa sub-Region because, right from inception, the CDA has been product of

innovative thinking involving renowned international scholars.

7
2.2 HISTORY

The Centre for Dryland Agriculture (CDA) is one of the newly established academic Centers

and Institutes in Bayero University. In February 2012 Bayero University won a competitive

grant from MacArthur Foundation to support the establishment and activities of the Centre in

the first three years. This support is under the MacArthur Higher Education Initiative in

Africa which is geographically focused in Nigeria and Madagascar, where the Foundation

works with leading Universities to strengthen them as Institutions and advance their

contributions to development of Africa. The Foundation hopes to build up University

Departments or Centres and integrate them into Graduate Training and Research Disciplinary

Networks involving other Universities and Institutions in Africa and beyond.

Bayero University established the CDA to be able to generate sustainable agricultural

technologies that would increase the productivity of crops and livestock in the semi-arid and

dry-sub-humid environments of Nigeria and the West African sub-region. The Centre would

build human resource capacity at the appropriate level to meet the demand for improved

technologies needed to adapt to the changing environment in the region. The Centre, as part

of its objectives, will support graduates who would be able to engage themselves in

production and research on dryland agriculture that will provide relevant and appropriate

solutions to the regional agricultural and development problems.

Thus, the CDA aims to be a regional center of excellence in teaching, research and

development in Agriculture, especially Dryland Agriculture. The Centre shall support degree

training at the postgraduate level, as well as specialized short-term trainings targeted at

imparting specific skills to specific clients. The postgraduate programme shall lead to the

award of Postgraduate Diploma (PGD), Master (M.Sc) and Doctorate (Ph.D) Degrees in Five

(5) areas of Dryland Agriculture as follows:

8
1 Natural Resource Management and Climate Change

2 Crops and Cropping Systems

3 Range and Livestock Management

4 Livelihoods and Natural Resource Economics

5 Mechanization in Agriculture

The priorities of the CDA are to create more effective multi-disciplinary and multi-

institutional research to strengthen capacities of the institutions and individuals. It is planned

that the research students supported by the CDA funds will derive the research agenda of the

Centre. The Center shall continue to write proposals, attract funds and fuel this process.

Collaboration with relevant Institutions and stakeholders is therefore key to the success of

Centre.

9
 2.3 Organogram of Center for Dryland Agriculture

CENTRE FOR DRYLAND AGRICULTURE

DIRECTOR ADMINISTRATORS

GIS LABORATORY DUPITY DIRECTOR

DUPITY DUPITY DIRECTOR  CDA FARM
TRAINING  SCREEEN
OUTREACH
DIRECTORS HOUSE

DUPITY DIRECTOR

RESEARCH

 TISSUE CULTURE LABORATORY

 WET CHEMISTRY LABORATORY
 CENTRAL LABORATORY
(INSTRUMENTAL)

10
2.4 VISION AND MISSION

VISION

To be a global Centre in dryland agriculture for excellent techniques, research and quality of

its product.

MISSION

To respond to the need of dry land region through eleventh high level human capacity

development and demand driven research contributing to food security, improved

Livelihoods and sustainable use of natural Resources.

11
 CHAPTER THREE

PROCESS, COMPONENTS AND DISCRIPTION

3.1 INTRODUCTION

Various activities have been carried out across three laboratories viz: tissue culture, wet

chemistry, and central laboratory. In tissue culture laboratory, general introduction of the

laboratory rules and regulations were given by the laboratory supervisor to ensure that it’s

free from any form of contaminations. After that, preparation for seed culture was carried out,

seeds and tissues were cultured. In the wet chemistry laboratory, soil physical and chemical

properties were analyzed using standard analytical procedure such as; pH, electrical

conductivity, particle size distribution, organic carbon, Mehlich 3 extraction etc. lastly, in the

central laboratory preparation of standards, centrifugation and elemental analysis were

carried out using Micro Plasma Atomic Emission Spectroscopy, centrifuge, ion

chromatography, CHNS analyzer, and near infra-red spectroscopy.

3.2 Tissue Culture Laboratory

The first week was mainly a general introduction to the lab and explanation of all the

laboratory rules and the regulations it being a lab that should always be free from

contaminants. During the second week, the search for indigenous seeds of the northern part

of Nigeria was the work assigned in preparation for seed culture. The seeds were soaked and

washed in the same week and were treated with their various controls in order to break their

dormancy. The third and fourth week was mainly focused on seed culture which involves

preparation of media, the choice of explants, surface sterilization and inoculation of the seeds.

Tissue culture was done in the fifth and sixth week which involves same procedure as seed

culture with the main difference being the choice of explant, and the type of media used for

inoculation. The remaining two weeks done at tissue culture lab involves sub culturing of

12
clones and inoculating them into a new culture media, transfer of plantlets into the soil, and

stem cutting of shoot in preparation for vegetative propagation. These were are the main

things done in plant tissue laboratory with the inclusion of minor works like watering of

seedlings, washing of glass wares and cleaning of the laminar flow and autoclave.

3.3 Wet Chemistry Laboratory

Determining of the soil pH was the next work in another different laboratory. The first two

weeks was introduction on different equipments, instruments and machines in the laboratory.

The science of soil pH and even determination of the soil pH of different samples of different

places together with the electronic conductivity of the soil sample was carried out, all in the

ninth and tenth week. The eleventh and twelfth week was the particle size distribution

experiment, where different soil samples were separated base on their sand, silt and clay

composition by addition of CALGON solution to separate the particles, and hydrometer

reading to determine the soil particles sedimentation rate.

After Sallah break intercepted in the thirteenth and fourteenth week, determination of organic

carbon present in the soil was achieved in the fifteenth and sixteenth week. It was determined

by the sulphuric acid and aqueous potassium dichromate (K 2Cr2O7) mixture. After complete

oxidation, the unused or residual K2Cr2O7 (in oxidation) is titrated against ferrous ammonium

sulphate. The used K2Cr2O7, the difference between added and residual K2Cr 2O7, gives a

measure of organic carbon content of soil. The last week in the wet chemistry laboratory was

committed to the extraction of elements by the Mehlich-3 solution. Different soil samples

were treated with the solution and then filtered, refrigerated or centrifuged and the run in the

MPAES machine in the central laboratory.

13
3.4 Central Laboratory (Instrumentation)

At the eighteenth week, the extracted samples from the previous weeks were run on MPAES

in the central laboratory. The samples were centrifuged and analyzed. The last weeks of the

SIWES programme were mainly covered with the elemental analysis in the central

laboratory. Different samples digestions were run on complicated machines in the laboratory

such as MP-AES. Multi-element calibration standards containing P, K, Ca, Mg and Si were

prepared in concentrations of 4, 5, 8, 10, 15, 16, 20, 40, 60 and 80 ppm.

14
 CHAPTER FOUR

EXPERIENCE ACQUIRED DURING THE EXERCISE

4.1 INTRODUCTION

This chapter gives a detail explanation of the work carried out during the SIWES program

and the overall experience gained including the training carried out, processes and principles.

Also contained in this chapter are some of the new equipments used which made the work

easier and more reliable.

4.2 TISSUE CULTURE

The term tissue culture may be defined as the process of in- vitro culture of explants (pieces

of living differentiated tissues) in nutrient medium under aseptic conditions. However, in

general, the tissue culture includes the term tissue culture as well as cell culture, organ culture

and suspension culture.

Plant tissue culture is not a separate branch of plant science like taxonomy, cytology, plant

physiology etc. Rather it is a collection of experimental methods of growing large number of

isolated cells or tissues under sterile and controlled conditions.

The cells or tissues are obtained from any part of the plant like stem, root, leaf etc. which are

encouraged to produce more cells in culture and to express their totipotency (i.e. their genetic

ability to produce more plants). Cells or tissues are grown in different types of glass vials

containing a medium with mineral nutrients, vitamins and phytohormones. Therefore, to

carry out the experiments using tissue culture techniques, a well-equipped laboratory is first

required.

4.2.1 TYPES OF PLANT TISSUE CULTURE

There are so many different types of tissue culture which include the following as follows
15
 i. Seed Culture

Seed culture is the type of tissue culture that is primarily used for plants such as orchids. For

this method, explants (tissue from the plant) are obtained from an in-vitro derived plant and

introduced in to an artificial environment, where they get to proliferate. In the event that a

plant material is used directly for this process, then it has to be sterilized to prevent tissue

damage and ensure optimum regeneration.

ii. Embryo Culture

Embryo culture is the type of tissue culture that involves the isolation of an embryo from a

given organism for in vitro growth. Note, the term embryo culture is used to refer to sexually

produced zygotic embryo culture. Embryo culture may involve the use of a mature or

immature embryo. Whereas mature embryos for culture are essentially obtained from ripe

seeds, immature embryo (embryo rescue) involves the use of immature embryos from

unripe/hybrid seeds that failed to germinate. In doing so, the embryo is ultimately able to

produce a viable plant.

iii. Callus Culture

Callus - This is the term used to refer to unspecialized, unorganized and a dividing mass of

cells. A callus is produced when explants (cells) are cultured in an appropriate medium - A

good example of this is the tumor tissue that grows out of the wounds of differentiated

tissues/organs. In practice, callus culture involves the growth of a callus (composed of

differentiated and non- differentiated cells), which is the followed by a procedure that induces

organ differentiation.

16
 iv. Organ Culture

Organ culture is a type of tissue culture that involves isolating an organ for in vitro growth.

Here, any organ plant can be used as explants for the culture process (Shoot, root, leaf, and

flower). With organ culture, or as is with their various tissue components, the method is used

for preserve their structure or functions, which allows the organ to still resemble and retain

the characteristics they would have in vivo. Here, new growth (differentiated structures)

continues given that the organ retains its physiological features. As such, an organ helps

provide information on patterns of growth, differentiation as well as development.

There are number of methods that can be used for organ culture. These include;

a) Plasma clot method - Here, the method involves the use of a clot that is composed of

plasma and chick embryo extract (or any other extract) in a watch glass.

b) Raft method - For this method, the explants is placed on a raft of lens paper/rayon acetate

and floated on a serum in a watch glass.

c) Agar gel method - The medium used for this method is composed of a salt solution, serum

as well as the embryo extract or a mixture of various amino acids and vitamin with 1 percent

agar. d) Grid method - Grid method, as the name suggests involves the use of perforated

stainless steel sheet, on which the tissue of interest is placed before being placed in a culture

chamber containing fluid medium.

v. Protoplast Culture

Protoplast -cells without cell walls. A protoplast is the term used to refer to cell (fungi,

bacteria, plant cells etc) in which the cell wall has been removed, which is why they are also

referred to as naked cells.

17
 vi. Cell Culture:

Denotes the in-vitro culture of single or a few cells.

vii. Suspension Culture:

Defined as the culture of cell and cell aggregates suspended in a liquid medium.

Explant: The excised piece of differentiated tissue or the organ which is used for culture is

called as explant. e.g., embryos, young leaf, bud, etc.

Callus: The undifferentiated mass of cells is referred to as callus. The cells of callus are

meristematic in nature.

4.2.2 PHASES OF PLANT TISSUE CULTURE

Initiation Phase (Stage 1)

The initiation phase is the first phase of tissue culture. Here, the tissue of interest is obtained,

introduced and sterilized in order to prevent any microorganism from negatively affecting the

process. It is during this stage that the tissue is initiated in to culture.

Multiplication Phase (Stage 2)

The multiplication phase is the second step of tissue culture where the in vitro plant material

is re- divided and then introduced in to the medium. Here, the medium is composed of

appropriate components for growth including regulators and nutrients. These are responsible

for the proliferation of the tissue and the production of multiple shoots.This step is often

repeated several times in order to obtain the desired number of plants.

18
Root formation (Stage 3)

It is at this phase that roots are formed. Here, hormones are required in order to induce

rooting, and consequently complete plantlets.

Hardening and Acclimatization of Tissue Culture Plantlets (stage 4)

This is the final stage and requires careful handling of plants. The transplantation from

completely controlled conditions should be gradual. This process of gradually preparing the

plants to survive in the field conditions is called acclimatization.

4.2.3 BASIC REQUIREMENTS OF PLANT TISSUE CULTURE

The main requirements of plant tissue culture are:

(1) Laboratory Organization

(2) Culture Media

(3) Aseptic Conditions

1. Laboratory Organization:

In a standard tissue culture lab, there must be a few basic facilities like:

i. A Media Room for preparation, sterilization and storage of culture media.

ii. Facilities for washing of lab- wares, explants, etc.

iii. Space for storage of lab-wares.

iv. Culture rooms or incubators where conditions of temperature, humidity and light etc. can

be maintained.

v. Observation and Data Collection area.

19
2. Culture Media:

The medium on which the explants are cultured is culture medium. It is composed of various

nutrients required for proper culturing. Culture media are largely responsible for the in vitro

growth and morphogenesis is of plant tissues. The success of the plant tissue culture depends

on the choice of the nutrient medium. In fact, the cells of most plant cells can be grown in

culture media.

Basically, the plant tissue culture media should contain the same nutrients as required by the

whole plant. It may be noted that plants in nature can synthesize their own food material.

However, plants growing in vitro are mainly heterotrophic i.e. they cannot synthesize their

own food. Different types of plants and organs need different compositions of culture media.

A number of media have been devised for specific tissues and organs. Some important of

them are:

 MS (Murashige and Skoog) Medium

 LS (Linsmaier and Skoog) Medium

 B5 (Gamborg’s) Medium

 White’s Medium, etc.

Important constituents of a culture medium are:

(i)Organic supplements:

(a) Vitamins like: thiamine (B), Pyridoxin (B), Nicotinic Acid (B), Etc.

(b)Antibiotics like: Streptomycin, Kanamycin;

(c) Amino Acids like: Arginine, Asparagine.

20
(ii) Inorganic Nutrients:

Micronutrients as Iron (Fe), Manganese (Mn), Zinc (Zn), Molybdenum (Mo), Copper (Cu),

Boron (B). Macronutrients include six major elements as Nitrogen (N), Sulphur (S),

Phosphorus (P), Potassium (K),Calcium (Ca), Magnesium (Mg).

(iii) Carbon and Energy Source:

Most preferred carbon source is Sucrose. Others include lactose, maltose, galactose,

raffinose, cellobiose, etc.

(iv) Growth Hormones:

a. Auxins-mainly for inducing cell division.

b. Cytokinins-mainly for modifying apical dominance and shoot differentiation.

c. Abscisic Acid (ABA)-Used occasionally.

d. Gibberellins-Used occasionally.

Gelling Agents: These are added to media to make them semisolid or solid. Agar, Gelatine,

Alginate etc. are common solidifying or gelling agents. Other Organic Extracts: Sometimes

culture media are supplemented with some organic extracts also like coconut milk, orange

juice, tomato juice, potato extract, etc. Stock Solution of MS Basal Medium 1 ltr of MS

medium = (50 ml of stock solution I)+ (5ml of each stock solutions II, III.IV)

4.2.4 PREPARATION AND STERILIZATION OF CULTURE MEDIA

A suitable culture medium is prepared with special attention towards the objectives of

culture and type of explants to be cultured. Prepared culture medium is transferred into

sterilized vessels and then sterilized in autoclave. The appropriate mixture (such as the MS

mixture) is mixed with distilled water and stirred while adding the appropriate amount of
21
sugar and sugar mixture. Here, sodium hydroxide or hydrochloric acid is used to adjust the

pH - Contents used here will depend on the plant to be cultured and the number of tissues to

be cultured. Agar is added to the mixture, heat and stirred to dissolve after cooling, the warm

medium is poured into glass jars (to a depth of about 4 cm) with lids sitting on the jars, the

jars are placed in a pressure cooker and sterilized for 20 minutes.

Composition of Media:

The composition of the culture media is primarily dependent on two parameters:

1. The particular species of the plant.

2. The type of material used for culture i.e. cells, tissues, organs, protoplasts.

Thus, the composition of a medium is formulated considering the specific requirements of a

given culture system. The media used may be solid (solid medium) or liquid (liquid medium)

in nature. The selection of solid or liquid medium is dependent on the better response of a

culture.

Synthetic and natural media:

When a medium is composed of chemically defined components, it is referred to as a

synthetic medium. On the other hand, if a medium contains chemically undefined compounds

(e.g., vegetable extract, fruit juice, plant extract), it is regarded as a natural medium.

Synthetic media have almost replaced the natural media for tissue culture.

Expression of concentrations in media:

The concentrations of inorganic and organic constituents in culture media are usually

expressed as mass values (mg/l or ppm or mg I ). However, as per the recommendations of

the International Association of Plant Physiology, the concentrations of macronutrients

should be expressed as mmol/l and micronutrients as µmol/l .

22
Constituents of Media:

Many elements are needed for plant nutrition and their physiological functions. Thus, these

elements have to be supplied in the culture medium to support adequate growth of cultures in

vitro.

Selection of a Suitable Medium:

In order to select a suitable medium for a particular plant culture system, it is customary to

start with a known medium (e.g. MS medium, B5 medium) and then develop a new medium

with the desired characteristics. Among the constituents of a medium, growth regulators

(auxins, cytokinins) are highly variable depending on the culture system. In practice, 3-5

different concentrations of growth regulators in different combinations are used and the best

among them are selected. For the selection of appropriate concentrations of minerals and

organic constituents in the medium, similar approach referred above, can be employed.

Medium-utmost Important for Culture: For tissue culture techniques, it is absolutely essential

that the medium preparation and composition are carefully followed. Any mistake in the

preparation of the medium is likely to do a great harm to the culture system as a whole.

3. Aseptic Conditions:

Maintenance of aseptic conditions is the most critical and difficult aspect of in-vitro

culturing experiments. Aseptic conditions mean the conditions free from any type of

microorganisms (so as to prevent the loss of experiment by contamination). For this,

sterilization (i.e., complete removal or killing of microbes) is done. The most common

contaminants in culture are fungi and bacteria. Measures to be taken for maintaining asepsis

during tissue culture are:

i. Sterilization of the culture vessels using detergents, autoclaves, etc.

23
ii. Sterilization of instruments like forceps, needles etc. by flame sterilization.

iii. Sterilization of culture medium using filter sterilization or autoclaving methods.

iv. Surface sterilization of explants using surface disinfectants like Silver Nitrate (1%), H O

(10-12%), Bromine water (1-2%), Sodium Hypochlorite solution (0.3-0.6%), etc.

The whole procedure of plant tissue culture is to be carried out essentially under aseptic

conditions. So, the overall design of the laboratory must focus on the maintenance of aseptic

conditions. Secondly, the worker is also required to have proper knowledge of operating

various equipments like: pH meter, balance, laminar air flow and microscope, etc. While

performing the tissue culture experiments there must present the first aid kits and fire

extinguishers in the laboratory to avoid any mishap or accident. In addition, proper attention

should be given while handling the toxic chemicals and all the chemicals should be kept in

correct labelled containers and bottles

4.2.5 GENARAL TECHNIQUES OF PLANT TISSUE CULTURE

General technique of plant cell, tissue and organ culture is almost the same with a little

variation for different plant materials. There are certain basic steps for the regeneration of a

complete plant from explants cultured on the nutrient medium. These basic steps for in-vitro

culturing of plants are:

1. selection and sterilization of explants

2. preparation of culture media

3. inoculation

4. incubation

5. sub culture

6. hardening

24
 1. SELECTION AND STERILIZATION OF EXPLANT

Suitable explants are selected and are then excised from the donor plant. Explants are then

sterilized using disinfectants. Explants can be steam, leave, buds and seeds. If it is the seeds

that were selected as explants the seeds dormancy should be break by using any method.

Cut the plant part in to small pieces (e.g. steam of sweat potato can be cut). On the other

hand, seeds can be used as a whole. Using detergent and water, wash the plant part or seed

for about 20 minutes. Transfer the plant part or seed in to sterilizing Clorox solution, shake

for a minute and leave to sock for 20 minutes. Using a lid, gently discard the Clorox and

retain the plant part or seed in the container. 70 percent alcohol should be used for the

sterilization of the equipment used and containers. Open the container and pour sterile water

to cover half the container, Cover with a sterile lid again and shake the container for 2 to 3

minutes in order to wash the tissue or seed and remove the bleach. Pour the water and repeat

this three times Using sterilized gloves, remove the plant part or seed from the container and

on to a sterile Petri dish. Using a sterile blade cut the plant material to smaller pieces of about

2 to 3 mm across avoiding the parts that have been damaged by bleach Using sterile forceps.

2. PREPARATION AND STERILIZATION OF CULTURE MEDIA

A suitable culture medium is prepared with special attention towards the objectives of culture

and type of explants to be cultured. Prepared culture medium is transferred into sterilized

vessels and then sterilized in autoclave. The general methodology for a medium preparation

involves preparation of stock solutions (in the range of 10x to 100x concentrations) using

high purity chemicals and demineralised water. The stock solutions can be stored (in glass or

plastic containers) frozen and used as and when required. Most of the growth regulators are

not soluble in water. They have to be dissolved in NaOH or alcohol.

25
Sterilization of Media:

The culture medium is usually sterilized in an autoclave at 121°C and 15 psi for 20 minutes.

Hormones and other heat sensitive organic compounds are filter-sterilized, and added to the

autoclaved medium.

3. INOCULATION

Sterilized explants are inoculated (transferred) on the culture medium under aseptic

conditions. Successful control of contamination largely depends upon the precautions taken

to prevent the entry of microorganisms at the time of transferring the sterilized explants on

the nutrient medium. Dust, hair, hands and clothes are the potential sources of contamination.

The inoculating chamber should be dust free the operator should wear sterile headgear and

clothes (aprons) before entering the culture area.

The hands should be wiped with 95% alcohol and the transfer area also should be cleaned and

wiped with 95% alcohol before starting the transfer process. Talking or sneezing should be

avoided during transfer of explants into the media. The neck or mouth of culture container

should be flamed, the transferring instruments also to be flamed and dipped in alcohol. Care

should be taken so that the explants should not touch the edge of culture vessel, and after

transferring, the mouth should be closed by cap or by cotton plug and petridishes to be sealed

by ‘Parafilm’. During transfer, it is also ensured that the plant tissue should be exposed to the

media properly.

4. INCUBATION OF CULTURE

Cultures are then incubated in the culture room where appropriate conditions of light,

temperature and humidity are provided for successful culturing. The tubes containing plant

26
sections may be placed in a well-lit area of the classroom although not in direct sunlight. The

shoots will probably grow more quickly if the explants are placed under fluorescent or

growlights to provide at least 12 hours of light per day. Ensure that the temperature does not

go over 28oC. New shoots should develop within 2 weeks, and should be well advanced in 3

to 4 weeks. Check the tubes daily and discard any that show signs of infection (before

discarding first sterilize in the pressure cooker or add bleach into the tube).

Roots can appear within 6 weeks. Cuttings will need to be moved into rooting media for roots

to properly develop. This transfer to the second, rooting media must be conducted under the

same sterile conditions as at the initiation of the culture. All necessary equipments should be

set up as before and properly sterilized. There will usually be several shoots that have arisen

from each explant. These shoots should be carefully separated by gently removing the whole

explant from the media with sterile forceps and then separating the shoots by gently pulling

them apart using two pairs of forceps. Each shoot should then be placed into a tube of rooting

media and the bottom of the shoot pushed into the media so that good contact is made. The

cap is replaced and the shoots are then allowed to grow as in step 1 until roots are formed,

usually within 2-3 weeks.

5. SUB CULTURE

Cultured cells are transferred to a fresh nutrient medium to obtain the plantlets. The growth

and development of tissues cultured in vitro are generally monitored by observing the

cultures at regular intervals in the culture room or incubators. Based on the observations

either with hand-lens or with the aid of simple microscope under aseptic conditions, the

explants may be required to transfer to new media (freshly prepared) or with new ingredients

or hormone composition depending on the state of growth of cell or tissue.

27
The same precautions and full aseptic conditions are maintained during the transfer process

also. The delaying of this process may lead to inhibition of proper development of tissues and

also delaying the regeneration of plantlets. In case of suspension culture the change of media

or fresh inoculation at quick intervals is needed and also for callus culture the sub-culturing

of the callus tissue is needed to get the callus tissue in dividing conditions.

6. HARDENING

The transplanting process can be a shock to rapidly growing seedlings especially when set out

into the cold windy garden in the spring. This is especially true for transplants started in the

greenhouse, cold frame, hotbed or home. These young seedlings can be made somewhat

resistant to heat, cold temperatures, drying and whipping winds, certain types of insect injury,

injury from blowing sand and soil particles and low soil moisture by a process termed

"hardening".

Method:

Any of the following can be used to harden transplants. A combination of all these techniques

at one time is more effective.

1. Gradually reduce water - water lightly at less frequent intervals but do not allow the plants

to wilt severely.

2. Expose plants to lower temperature than is reported as optimal for their growth. If

biennials are exposed to cold for an extended period, they may bolt in lieu of developing

properly.

Note: Placing the plants outside during the day to encourage hardening and then bringing the

plants back into the warm house during the night often reverses the hardening process. Plants

28
could be placed in a cold frame or other area that does not freeze during the night hours

without lose of the hardening process.

3. Do not fertilize, particularly with nitrogen immediately before or during the hardening

process. A starter solution or liquid fertilizer could however be applied to the hardened

transplants one or two days prior to transplanting into the garden or at the time of

transplanting.

4. Gradually expose the plants to more sunlight. This results in the development of a thicker

cuticle layer thereby reducing water loss.

Cautions:

Hardening is not necessary for all transplants. Overly hardened plants while withstanding

unfavorable outside conditions are slow to get started and may never overcome the stress

placed on the plant during the hardening process.

After the hardening process (i.e., acclimatization of plantlet to the environment), the plantlets

are transferred to green house or in pots.

Transplantation of the Regenerated Plant:

Plants regenerated from in vitro tissue culture are transplanted to soil in pots. Prior to transfer

to pots, the acclimatization of these regenerated plants are needed. The plants at this time

develop adequate root systems and cuticular leaf surface structure so that it can withstand the

field environmental condition. The process of acclimatization needs the humid chamber and a

slow process to make the plantlet habituated from high humid condition to normal

atmospheric humidity. The greenhouse or the growth chamber should have artificial light

system also which includes a mixture of fluorescent and incandescent lamps designed to

provide balanced wavelengths of light for plant growth and photosynthesis.

29
The greenhouse facilities are needed for winter crops and summer crops differently for

maintenance of proper temp, required, air circulation and the relative humidity. The potted

plants are grown in field for further observation, flowering and normal seed setting to get the

next progeny.

4.3 WET CHEMISTRY

Analyzing the chemical characteristics of soils and plants in a reliable, broadly applicable,

cost-and time effective manner is of great importance to the agricultural, environmental and

development communities.

Soil nutrient depletion in small hold farming systems became recognized as a causal force

leading to chronic food insecurity and rural poverty. This awareness led to greater emphasis

upon examining nutrient cycles and budgets at scales ranging from the farm to the regional

levels. This information in turn contributed to our abilities to develop and implement

strategies for soil fertility replenishment. Recent developments signal the need to better

analyze the chemical compositions of plants and soil. Plant breeders no longer select crops

based upon yield properties alone, but rather recognize the importance of nutrient use

efficiency and tolerance to nutrient stress.

Soil biology is now sufficiently developed that litter decomposition and nutrient

mineralization operate in a more predictable manner allowing the benefits from organic

inputs to be better managed. Computer simulation models have become an important tool for

the integration and extrapolation of research results, but these models require careful

initialization and validation with carefully collected data before the outputs obtain credibility.

30
4.3.1 SOIL pH AND ELECTROCONDUCTIVITY

Soil pH is a measurement of the acidity or alkalinity of a soil. pH is define as the negative

logarithm of hydrogen ions of the soil. On the pH scale, 7.0 is neutral, below 7.0 is acidic and

above 7.0 is alkaline. Soil pH is important because it can affect the availability of plant

nutrients and as well as the soil ecology. pH meter is used in the determination of the pH.

Measurement of pH is expressed as the inverse log of the hydrogen ion concentration. The

pH of the soil solution controls the form and solubility of many plant nutrients. Soil pH is

measured on 1:1 soil water suspension. The electroconductivity measurement identifies soils

which are potentially saline. The electroconductivity of the saturated paste extract is

measured to determine the level of salinity. Soil electroconductivity is measured on 2.5:1 soil

water suspension.

MATERIALS AND REAGENTS

Materials: glass rod, Glass-electrode pH meter, Sample cups, beaker, Weighing boat,

Spatula, Wash bottle, Conductivity meter.

Reagent: Distilled water.

The only reagent we used was distilled water because we needed to determine the soil pH

in water.

PROCEDURE:

Weigh 10g of air-dry soil (passed 2mm sieve) into a small cup, Add 10ml of distilled water

and allow standing for 30min and stirring occasionally with glass rod. Insert the electrodes of

the pH meter into the partly settled suspension and measure the pH. Do not stir the

suspension during measurement. Add 15ml of distilled water into the suspension and stirred.

Insert the electrode of the electro conductivity meter into the suspension and measure the

31
electro conductivity. The electrodes should be rinsed with deionized water and wiped dry

with a clean tissue or filter paper after each reading. The pH meter should be calibrated with

pH 7.0 and pH 4.0 buffers before use.

4.3.2 SOIL PARTICLE SIZE ANALYSIS

The particle size analysis of a soil estimates the percentage of sand, silt and clay contents of

the soil and is often reported as percentage by weight of oven-dry and organic matter-free

soil. The analyses are usually performed on air-dry soil. Based on the proportions of

different particle sizes, a soil textural category may be assigned to the sample. The first stage

in a particle size analysis is the dispersion of the soil into the individual particles. These are

the sand (2.00 - 0.05 mm), silt (0.05 - 0.002 mm) and clay (< 0.002 mm) fractions.

Individual soil particles are often bound into aggregates hence the requirement for dispersion.

The hydrometer method of silt and clay measurement relies in the effects of particle size on

the differential settling velocities within a water column. The settling velocity is also a

function of liquid temperature, viscosity and specific gravity of the falling particle.

Theoretically the particles are assumed to be spherical and to have a specific gravity of 2.65.

If all other factors are constant then the settling velocity is proportional to the square of the

radius of the particle (Stoke's law). In practice, therefore, we must know and make correction

for the temperature of the liquid. Greater temperatures result in reduced viscosity due to

liquid expansion and a more rapid descent of falling particles.

Apparatus: Weighing balance, Weighing boat, Spatula, 1000ml measuring cylinder,

Thermometer, Stop watch, Sample bottles, Hydrometer, Soil dispersing plunger,

Reciprocating shaker.

Reagents: Calgon Solution: Add 35.7g sodium hexameta phosphate and 7.94g NACO Into

distilled water of 1 litre.

32
PROCEDURE:

Weigh 51.0gAirdried soil sample sieved through 2mm sieve into a plastic bottle, add 50ml of

carbon solution into each sample, add 10ml of de-ionized water into each sample and stir,

Shake for 30minutes. Then transfer into cylinder, Add up de-ionized water to mate to volume

of 1litre. Measure temperature T1 through thermometer, mix the sample with plunger up to

ten times. Drop the hydrometer immediately you remove the plunger and wait for 40 seconds

for the reading H1, wait for 3 hours for the second reading (H2 and T2).

To determine the distribution of sand, silt and clay, calculations are made. To calculate sand

as (PERCENTAGE) %: after 40 seconds, the sand has settled and the hydrometer reading

reflects the grams of silt + clay in l litre of the suspension. To calculate the amount of sand

present in 1 litre of the suspension, subtract this value from the original sample weight.

Sample Calculation: If the hydrometer reading after 40 seconds corrected for temperature is

18.0 g per litre, then silt + clay weigh 18.0 g in the 1 litre soil suspension. Therefore, the

sand weighs 51.0 - 18.0 = 33.0 g in the 1 litre suspension (of the original 51.0 g of air-dry

soil sample). The percentage of sand is calculated by dividing the sand content (33 g) by the

total (51 g) and multiplying by 100 as follows:

sand % = (33.0 / 51.0) x 100 = 64.7%

Clay: After 3 hours, the silt has settled. The hydrometer reading now reflects the clay content

of the original suspension. For example, if hydrometer reading after temperature correction

is 4.7 g/litre, then the percentage of clay in soil is:

clay % = (4.7 / 51) x 100 = 9.2%

Silt: The silt content is calculated by subtracting the sum of the clay and sand contents from

100%

33
 silt % = 100 - (9.2% clay + 64.7% sand) = 26.1%

The assumption made here is that the organic matter is negligible. However, if soil is found to

be high, treat the soil with hydrogen peroxide until the frothing reaction subsides. An

alternatively, determine the organic matter content and subtract it from 100 before assigning

it in the formulae.

4.3.3 ORGANIC CARBON CONTENT OF SOILS

Organic carbon is determined by the sulphuric acid and aqueous potassium dichromate

(K2Cr2O7) mixture. After complete oxidation, the unused or residual K 2Cr2O7 (in oxidation)

is titrated against ferrous ammonium sulphate. The used K2Cr2O7, the difference between

added and residual K2Cr2O7, gives a measure of organic C content of soil.

Apparatus: Weighing balance, Spatula, Weighing boat, Conical flask, Beaker, Magnetic

stirrer, Retort stand, Burette, Dropper, funnel.

Solutions

 Potassium dichromate solution: Dissolve 49.024 g of dry K2Cr2O7 in 800 ml of

distilled water, and dilute to 1000 ml.

 Ferrous ammonium sulphate solution. Dissolve 78.390 g ferrous ammonium sulphate

in 50 ml conc. H2SO4, and dilute to 1000 ml with distilled water.

 Indicator solution: Dissolve 1.485 g of phenanthroline in 100 ml of 0.025 M ferrous

sulphate (0.695 g of ferrous sulphate, FeSO4.7H2O) in 100 ml of distilled water.

 Distilled water.

Weigh 0.5 g of Airdried soil sample (sieved through 2mm sieve) into a conical flask, add 5

ml of K2Cr2O7, 10 ml of H2SO4 and allow resting for 30 minutes. Add 100 ml of distilled

water apply indicator and tittered against FeSO4. Take the initial and final burette reading.
34
Calculations:

Organic Carbon (%) = (B – A) * [(5 ∕B) * (0.3)] ∕ W

Organic Matter = Organic Carbon * 1.72

Where B = blank, A = actual, W = weigh of sample.

4.3.4 Mehlich-3 extraction and Analysis

Mehlich-3 procedure is amongst the most popular multi element soil extraction procedures.

Apart from extracting elements in a single extraction, it requires only 15 minutes of total

extraction time, followed by either filtration or centrifugation. For soils with pH <7.5, it gives

virtually identical results for Ca, Mg, and K as the ammonium acetate extraction, and is very

highly correlated with other cation extraction procedures. The extractant is a buffered acid

matrix, which is adversely affected by soils with free carbonates which neutralize the

extractant. Therefore, it is not appropriate with pH 7.5 and above.

Apparatus: weighing boat, spatula, Pippettes, reciprocating shaker, beaker, Plastic vials,

sample bottle, Funnel, Filter paper, Centrifuge, MP-AES.

Reagents: Distilled water, Ammonium fluoride (Na4F), Ethylenediamine tetra acetic acid

(EDTA), Ammonium nitrate (NH4NO3), Acetic acid, Nitric acid

Standard solutions: 1000ppm for Ca, Mg, K, P, Cu, Zn, Mn and Fe

Mehlich-3 extraction solution: Dissolve 138.9g of Na4F in 600 ml of distilled water, add

73.06g of EDTA, stirred and filled up to 1L (Stock). Dissolve 80.05g of NH4NO3 in 3 liters

of distilled water; add 16 ml of your stock solution above. Add 46 ml of CH 3COOH and 5 ml

of HNO3, set the pH to 2.5 and top the solution to 4 liters.

35
Extraction: Weigh 6.0 g of dried soil into a plastic bottle. Add 60 ml of Mehlich-3 solution

and shake for 5 minutes. Filter it and send it to the centrifugation machine for centrifuging for

10 minutes at 5000 rpm. The supernatant is the transferred to a test tube and taken to the

Micro Plasma Atomic Emission Spectroscopy machine MPAES including the standard

solutions machine for Ca, Mg, Na and K analysis.

4.4 Central Laboratory (Instrumentation)

Appropriate plant nutrition is crucial to plant health and for adequate growth of the crops

with maximal productivity. Among the nutrients consumed by the plants, N, P, K, Ca, Mg

and S are essential, required in large quantities, while B, Cl, Co, Cu, Fe, Mn, Mo and Si are

needed at lower levels. The determination of macro and micronutrients in plant tissues is an

important measure used to analyze plant nutritional status and to evaluate the possible need

for fertilizer supplementation. The analysis of leaves provides the most information for the

deficiency or excess of nutrients in plants.

Accurate elemental analysis of soils is also extremely important for numerous reasons. Lead

and cadmium are known for their adverse health and environmental effects. P, Cu, Fe, Mg are

considered important macro and micro nutrients for plants and thus, these elements are of

interest for agricultural science, crop selection and soil remediation studies. Titanium

minerals in soil are also used as weathering indicators.

4.4.1 Determination of macro and micronutrients in plants

Instrumentation

All measurements were carried out using the Agilent 4200 MP-AES fitted with the Agilent

4107 Nitrogen Generator. The sample introduction system consisted of a double pass

cyclonic spray chamber, OneNeb nebulizer, Solvaflex pump tube (orange/green) and Easy-fit

36
torch to introduce the sample. The intuitive MP Expert software features easy to use auto-

optimization tools, which allow for quick optimization of the nebulization gas flow rate and

viewing position for each element and wavelength in the method. In addition, MP Expert

software allows for automatic background correction.

Standard and sample preparation

Multi-element calibration standards containing P, K, Na, Ca, Mg and Si were prepared in

concentrations of different ppm, for macro and micronutrients determination. Corn plant

samples were analyzed. Samples were microwave-assisted acid digested by adding 6 ml of

HNO3 and 4 ml H2O2 to 0.2 g of material. All samples were microwave digested using the

microwave digester.

Calibration linearity

The calibration curves obtained for macronutrients and micronutrients showed good linearity

across the concentration range. Dynamic linear range was 0-4ppm for all micronutrients and

0 – 75ppm for all macronutrients analyzed. All calibration curves were obtained using linear

calibration fit with the exception of K, for which a rational fit was used.

4.5 PROBLEMS ENCOUNTERED

During the course of the programme, problems were encountered. Problems like that of risk

of contamination, poor data collection and instrument/machine failure were common. Below

were the main problems encountered

 Absence of green house near the tissue culture laboratory.

 Some works in the laboratories are left to spoil due to poor supervision.

 Re-use of disposable materials due to the economic problems

37
These problems all together can be mitigated by dedication, technical know-how, financial

support and zeal.

4.6 PROBLEMS SOLVED

Conducting experiments involves risk of getting spoiled. Some of the problems that spoil

experiments are solved by good thinking. Some of the problems solved during the

programme include:

 In the tissue culture laboratory, a growth media was prepared for spraying on

seedlings that are wilting to regain it form.

 When preparing the solution of mehlich-3 extraction, the pH fall below 2.5 and

NH4OH was added to adjust the pH to normal.

38
 CHAPTER FIVE

SUMMARY, RECOMMENDATION AND CONCLUSION

5.1 INTRODUCTION

Students industrial work experience scheme (SIWES) is very interesting course, which helps

student to understand what they are thought in the classes by conducting practicals in the

industries and/or organization they attend. It gives chances to the students to learn about the

working environment and to have working experiences.

5.2 SUMMARY

The term tissue culture may be defined as the process of in- vitro culture of explants (pieces

of living differentiated tissues) in nutrient medium under aseptic conditions. However, in

general, the tissue culture includes the term tissue culture as well as cell culture, organ culture

and suspension culture. Tissue culture involved the selection and sterilization of explants,

preparation of culture media, inoculation, incubation and hardening processes under aseptic

conditions.

Analyzing the chemical characteristics of soils and plants in a reliable, broadly applicable,

cost-and time effective manner is of great importance to the agricultural, environmental and

development communities. Several experiments to determine the physicals and chemical

properties of the soil samples such as the pH determination, particle distribution, and organic

carbon content gives broad and vital information. This information in turn contributed to our

abilities to develop and implement strategies for soil fertility replenishment. Recent

developments signal the need to better analyze the chemical compositions of plants and soil.

Plant breeders no longer select crops based upon yield properties alone, but rather recognize

the importance of nutrient use efficiency and tolerance to nutrient stress.

39
5.3 RECOMMENDATION

The student Industrial Work Experience Scheme (SIWES) is an interesting and

knowledgeable method of providing undergraduate student of high institution, students of

Colleges and Polytechnics with enough practical exposure. The following recommendations

are hereby made to whom it may refer:

 The government and stakeholders should pay more attention to this kind of issues by

providing financial support and working facilities.

 The centre should provide green house near the tissue culture laboratory so as to ease

more lost, damage of the culture seedlings.

 Yusuf maitama sule university should establish a centre like CDA to produce highly

skilled manpower and research products that will address the challenges of

agricultural development.

5.4 CONCLUSION

The SIWES program I underwent at CDA, BUK, Kano, gave me the opportunity to learn and

appreciate my field of study as well as laboratory research practices. The Centre for Dryland

Agriculture (CDA) is a regional centre of excellence established to produce highly skilled

manpower and research products that will address the challenges of agricultural development

within the dryland areas of West and Central Africa (WCA) and expand the capacity of the

region to deal with issues of food security, natural resources management, climate change,

livelihoods and broader development challenges.

The scheme also exposed me to real life working environment, enabling me to put into

practice of what I have learnt in the school, made me understood the technical application of

profession as well as competence, standard and professionalism so that I can readily

40
contribute to the fast growing economy of the country, as well as inoculation of my attitude

of punctuality and hard working.

The SIWES programme had been a major breakthrough for me in my academic pursuit as a

Botany student not only because it unveiled so many phenomena which remained a mystery

to me but because it also exposed me to the real challenges that are ahead of me as a Botany

student. During this program, I became acquainted with most equipment and processes

involved in the various laboratory activities; also I was able to learn the use of techniques in

Tissue Culture, their steps, stages, advantages and disadvantages.

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 REFERENCES

Clayton G. L. et al (2017). Determination of macro and micronutrients in plants using the

Agilent 4200 MP-AES. Brazil: Agilant Technologies Inc.

Dave M. and Alejandro A., (2015). Direct determination of Cu, Fe, Mn, P, Pb and Ti in HF

acid-digested soils using the Agilent 4200 Microwave PlasmaAtomic Emission

Spectrometer. Australia: Agilent Technologies Melbourne.

Okalebo J. R., Gathua W. K., & Woomer P. L. (2002). Laboratory Methods of Soil and

Plants Analysis: A Working Manual. Kenya: TSBF-CIAT and sacred Africa.

Sharma K. G., Jagetiya S., & Dashora R. (2015). General Techniques of Plant Tissue Culture.

United State: Lulu Press Inc.

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